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ABSTRACT O ‐Methylation catalyzed by plant O ‐methyltransferase plays a crucial role in both drug design and biosynthesis of natural products. However, their practical applications are often restricted by strict substrate specificity and a strong dependence on the expensive methyl donor S‐adenosyl‐L‐methionine (SAM). Herein, an O ‐methyltransferase, SmOMT, is identified from the medicinal plant Selaginella moellendorffii , exhibiting substrate promiscuity and regioselectivity. SmOMT catalyzed the methylation of 25 structurally diverse substrates and demonstrated detectable N ‐methylation activity. Combined ternary complex structure and molecular dynamics studies of SmOMT elucidate its catalytic and regioselectivity mechanisms. A double mutant, SmOMT M2 , with enhanced catalytic activity is obtained based on structural analysis. To overcome SAM dependence, a cascade system for SAM regeneration is successfully constructed by coupling SmOMT M2 with a mutant halide methyltransferase, AtHMT V140T . Employing the iMARS platform, a highly active fusion enzyme, AtHMT V140T ‐L 95 ‐SmOMT M2 , is designed. This fusion enzyme outperforms the free‐enzyme cascade system and facilitates the gram‐scale synthesis of a series of methylated compounds with enhanced anti‐inflammatory activity. This work provides a versatile methylating biocatalyst and establishes an efficient SAM regeneration methylation platform, overcoming limitations in enzymatic methylation and enabling the sustainable production of high‐value pharmaceuticals.
Xiong et al. (Fri,) studied this question.